Directional genomic hybridization: inversions as a potential biodosimeter for retrospective radiation exposure.

Chromosome aberrations in blood lymphocytes provide a useful measure of past exposure to ionizing radiation. Despite the widespread and successful use of the dicentric assay for retrospective biodosimetry, the approach suffers substantial drawbacks, including the fact that dicentrics in circulating blood have a rather short half-life (roughly 1-2 years by most estimates). So-called symmetrical aberrations such as translocations are far more stable in that regard, but their high background frequency, which increases with age, also makes them less than ideal for biodosimetry. We developed a cytogenetic assay for potential use in retrospective biodosimetry that is based on the detection of chromosomal inversions, another symmetrical aberration whose transmissibility (stability) is also ostensibly high. Many of the well-known difficulties associated with inversion detection were circumvented through the use of directional genomic hybridization, a method of molecular cytogenetics that is less labor intensive and better able to detect small chromosomal inversions than other currently available approaches. Here, we report the dose-dependent induction of inversions following exposure to radiations with vastly different ionization densities [i.e., linear energy transfer (LET)]. Our results show a dramatic dose-dependent difference in the yields of inversions induced by low-LET gamma rays, as compared to more damaging high-LET charged particles similar to those encountered in deep space.

Association of inter- and intrachromosomal exchanges with the distribution of low- and high-LET radiation-induced breaks in chromosomes.

To study the effects of low- and high-linear energy transfer (LET) radiation on break locations within a chromosome, we exposed human epithelial cells in vitro to (137)Cs γ rays at both low and high dose rates, secondary neutrons at a low dose rate, and 600 MeV/u iron ions at a high dose rate. Breakpoints were identified using multicolor banding in situ hybridization (mBAND), which paints chromosome 3 in 23 different colored bands. For all four radiation scenarios, breakpoint distributions were found to be different from the predicted distribution based on band width. Detailed analysis of chromosome fragment ends involved in inter- or intrachromosomal exchanges revealed that the distributions of fragment ends participating in interchromosomal exchanges were similar between the two low-LET radiation dose rates and between the two high-LET radiation types, but the distributions were less similar between low- and high-LET radiations. For fragment ends participating in intrachromosomal exchanges, the distributions for all four radiation scenarios were similar, with clusters of breaks found in three regions. Analysis of the locations of the two fragment ends in chromosome 3 that joined to form an intrachromosomal exchange demonstrated that two breaks with a greater genomic separation can be more likely to rejoin than two closer breaks, indicating that chromatin folding can play an important role in the rejoining of chromosome breaks. Comparison of the breakpoint distributions to the distributions of genes indicated that the gene-rich regions do not necessarily contain more breaks. In general, breakpoint distributions depend on whether a chromosome fragment joins with another fragment in the same chromosome or with a fragment from a different chromosome.

mBAND analysis of chromosome aberrations in human epithelial cells induced by gamma-rays and secondary neutrons of low dose rate.

Human risks from chronic exposures to both low- and high-LET radiation are of intensive research interest in recent years. In the present study, human epithelial cells were exposed in vitro to gamma-rays at a dose rate of 17 mGy/h or secondary neutrons of 25 mGy/h. The secondary neutrons have a broad energy spectrum that simulates the Earth's atmosphere at high altitude, as well as the environment inside spacecrafts like the Russian MIR station and the International Space Station (ISS). Chromosome aberrations in the exposed cells were analyzed using the multicolor banding in situ hybridization (mBAND) technique with chromosome 3 painted in 23 colored bands that allows identification of both inter- and intrachromosome exchanges including inversions. Comparison of present dose responses between gamma-rays and neutron irradiations for the fraction of cells with damaged chromosome 3 yielded a relative biological effectiveness (RBE) value of 26+/-4 for the secondary neutrons. Our results also revealed that secondary neutrons of low dose rate induced a higher fraction of intrachromosome exchanges than gamma-rays, but the fractions of inversions observed between these two radiation types were indistinguishable. Similar to the previous findings after acute radiation exposures, most of the inversions observed in the present study were accompanied by other aberrations. The fractions of complex type aberrations and of unrejoined chromosomal breakages were also found to be higher in the neutron-exposed cells than after gamma-rays. We further analyzed the location of the breaks involved in chromosome aberrations along chromosome 3, and observed hot spots after gamma-ray, but not neutron, exposures.

Baseline and treatment-induced chromosomal abnormalities in peripheral blood lymphocytes of Hodgkin's lymphoma patients.

PURPOSE: To study chromosomal abnormalities in 49 patients with Hodgkin's lymphoma (HL), before and after treatment and at several times during a 2-year period. METHODS AND MATERIALS: Simple chromosomal aberrations (CAs) and complex chromosomal rearrangements (CCRs) were counted in peripheral lymphocytes by painting of chromosomes 1, 3, and 4 (fluorescence in situ hybridization). A control population was composed of 20 healthy donors and 69 untreated cancer patients who had undergone various radiologic scans. RESULTS: A greater frequency (p < 10(-4)) of spontaneous cytogenetic abnormalities was observed in untreated HL patients compared with the control populations. CCRs were observed exclusively in the HL population (p < 10(-4)). Chemotherapy was associated with a significant increase in the frequency of CAs (p < 10(-4)), according to the chemotherapy regimen (p = 0.002). Immediately after radiotherapy, a significant increase (p < 10(-4)) was observed in CAs according to the size of the irradiation field. Conversely, the significant increases in the frequency of CCRs observed after treatment did not correlate with the chemotherapy regimens, radiotherapy dose, or size of the irradiation field. The evolution of CAs vs. CCRs over time was also dissociated: during the follow-up of these patients, a significant decrease was observed in the frequency of CAs at 6 months and 1 and 2 years. In contrast, after an initial decrease for up to 6 months after treatment, the frequency of CCRs remained constant for up to 2 years. CONCLUSION: Increased cytogenetic abnormalities were observed in untreated HL patients compared with the control populations. The greater frequency of cytogenetic abnormalities persisted in some patients. The presence of CCRs supports the concept of a unique genetic environment in HL patients that persists in response to potentially noxious treatments.

Premature chromosome condensation associated with fluorescence in situ hybridisation detects cytogenetic abnormalities after a CT scan: evaluaton of the low-dose effect.

The purpose of this study was to assess the cytogenetic effects of the X ray irradiation used during a CT scan in order to estimate the mean absorbed dose in circulating lymphocytes. Chromosomal aberrations were scored in blood lymphocytes of ten patients undergoing CT scans, by applying fluorescence in situ hybridisation (FISH) to metaphase cells and premature chromosome condensation (PCC) with chromosomes 1, 3 and 4 painting probes immediately after exposure. This generated a dosimetric index that reflects the dose to the circulating lymphocytes. By using PCC a significant increase in the frequency of chromosomal fragment was observed immediately after a CT scan. However, no significant increase in chromosomal aberration was detected in metaphase cells. The mean dosimetric index immediately after exposure was 0.057 Gy (95% CI: 0.052-0.082 Gy). This dosimetric index depends essentially on the size of the examined and exposed blood volumes. This dose is in close agreement with the dose length product (DLP) (Gy cm) (R = 0.80). It should be kept in mind when justifying requests for diagnostic CT scan especially in young patients. The presence of chromosomal fragments after a CT scan indicated the cytogenetic effect of a low dose. PCC associated with chromosome painting is a method for detecting the cytogenetic effect of a low dose immediately after exposure.

Gene amplification and microsatellite instability induced in tumorigenic human bronchial epithelial cells by alpha particles and heavy ions.

Gene amplification and microsatellite alteration are useful markers of genomic instability in tumor and transformed cell lines. It has been suggested that genomic instability contributes to the progression of tumorigenesis by accumulating genetic changes. In this study, amplification of the carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase (CAD) gene in transformed and tumorigenic human bronchial epithelial (BEP2D) cells induced by either alpha particles or (56)Fe ions was assessed by measuring resistance to N-(phosphonacetyl)-l-aspartate (PALA). In addition, alterations of microsatellite loci located on chromosomes 3p and 18q were analyzed in a series of primary and secondary tumor cell lines generated in nude mice. The frequency of PALA-resistant colonies was 1-3 x 10(-3) in tumor cell lines, 5-8 x 10(-5) in transformed cells prior to inoculation into nude mice, and less than 10(-7) in control BEP2D cells. Microsatellite alterations were detected in all 11 tumor cell lines examined at the following loci: D18S34, D18S363, D18S877, D3S1038 and D3S1607. No significant difference in either PALA resistance or microsatellite instability was found in tumor cell lines that were induced by alpha particles compared to those induced by (56)Fe ions.

Evaluating changes in stable chromosomal translocation frequency in patients receiving radioimmunotherapy.

PURPOSE: The lack of any consistent correlation between radioimmunotherapy (RIT) dose and observed hematologic toxicity has made it difficult to validate RIT radiation dose estimates to marrow. Stable chromosomal translocations (SCT) which result after radiation exposure may be a biologic parameter that more closely correlates with RIT radiation dose. Increases in the frequency of SCT are observed after radiation exposure and are highly correlated with absorbed radiation dose. SCT are cumulative after multiple radiation doses and conserved through an extended number of cell divisions. The purpose of this study was to evaluate whether increases in SCT frequency were detectable in peripheral lymphocytes after RIT and whether the magnitude of these increases correlated with estimated radiation dose to marrow and whole body. METHODS AND MATERIALS: Patients entered in a Phase I dose escalation therapy trial each received 1-3 intravenous cycles of the radiolabeled anti- carcinoembryonic antigen (CEA) monoclonal antibody, 90Y-chimeric T84.66. Five mCi of 111In-chimeric T84.66 was co-administered for imaging and biodistribution purposes. Blood samples were collected immediately prior to the start of therapy and 5-6 weeks after each therapy cycle. Peripheral lymphocytes were harvested after 72 hours of phytohemagglutinin stimulation and metaphase spreads prepared. Spreads were then stained by fluorescence in situ hybridization (FISH) using commercially available chromosome paint probes to chromosomes 3 and 4. Approximately 1000 spreads were evaluated for each chromosome sample. Red marrow radiation doses were estimated using the AAPM algorithm and blood clearance curves. RESULTS: Eighteen patients were studied, each receiving at least one cycle of therapy ranging from 5-22 mCi/m2. Three patients received 2 cycles and two patients received 3 cycles of therapy. Cumulative estimated marrow doses ranged from 9.2 to 310 cGy. Increases in SCT frequencies were observed after each cycle for both chromosomes 3 and 4 in 16 of 18 patients and in at least one chromosome for the remaining 2 patients. Cumulative increases in SCT frequencies ranged from 0.001 to 0.046 with no major differences observed between chromosomes 3 and 4. A linear correlation between cumulative marrow dose and increases in SCT frequencies was observed for chromosome 3 (R2 = 0.63) and chromosome 4 (R2 = 0.80). A linear correlation was also observed between increases in SCT frequency and whole body radiation dose or administered activity (R2 = 0.67-0.89). There was less correlation between observed decrease in wbc or platelet counts and marrow dose, whole body dose, or administered activity (R2 = 0.28-0.43). CONCLUSIONS: Increases in SCT frequency were detectable in peripheral lymphocytes after low dose-rate RIT irradiation. A linear correlation was observed between increases in SCT and marrow dose, whole body dose, and administered activity. This correlation provides one of the strongest radiation dose-response and activity-response relationships observed with RIT. The detection of SCT may therefore have application as an in situ integrating biodosimeter after RIT. This biologic parameter should prove useful in comparing effects on marrow for different therapeutic radionuclides and in comparing effects of RIT and external beam radiation doses on a cGy per cGy basis. As a result, this should allow for a more direct comparison between different methods of irradiation and in further refinement of radioimmunotherapy dose estimates and dosimetry methodology.

Radiation-associated sarcomas are characterized by complex karyotypes with frequent rearrangements of chromosome arm 3p.

Ionizing radiation is a well-known risk factor for sarcoma development. To investigate whether radiation-associated sarcomas are characterized by chromosome aberrations that distinguish them from de novo sarcomas, we identified those patients in our series of more than 500 cytogenetically abnormal sarcomas that fulfilled the following criteria: (1) each patient should have been irradiated for another malignancy at least 3 years prior to the sarcoma diagnosis, and (2) the sarcoma should have developed within the field of radiation. Ten patients fulfilling these criteria could be retrieved (median age at sarcoma diagnosis was 55 years, range 17-79; median latency period between primary tumor and radiation-associated sarcoma was 9 years, range 4-30). The diagnoses were typical for radiation-associated sarcomas: 2 each of malignant fibrous histiocytoma, leiomyosarcoma, and pleomorphic sarcoma, and 1 each of osteosarcoma, fibrosarcoma, myxofibrosarcoma, and spindle cell sarcoma. All 10 cases had relatively complex karyotypes with multiple, mostly unbalanced, structural rearrangements, similar to what has been reported in de novo sarcomas of the corresponding histologic subtypes. The only cytogenetic features that were unusually frequent among the radiation-associated sarcomas were the finding of unrelated clones in 3 cases, and loss of material from chromosome arm 3p, in particular 3p21-3pter, in 8 cases. Loss of the same chromosome segment has been described in 4 of the 8 previously published cases of radiation-associated sarcomas that have been analyzed after short-term culturing, which makes this imbalance significantly (P < 0.001) more frequent among radiation-associated sarcomas (12 of 18 cases) than among unselected cases of the corresponding histologic subtypes (74 of 282 cases). In contrast to the cytogenetic results, no 3p deletions were detected among the 6 cases of the present series that could be analyzed by comparative genomic hybridization (CGH). The most frequent imbalance detected by CGH was gain of 15cen-q15 (3 cases), followed by loss of chromosome 13 and gain of 5p, and 7cen-q22, each detected in 2 cases.

Radiation fusion hybrids for human chromosomes 3 and X generated at various irradiation doses.

We have used a gamma-irradiation (2.5-25 krads) cell fusion procedure to generate human-hamster somatic cell hybrids (IHB, irradiated human fragments in B14-150 cells), retaining small fragments derived from human chromosomes 3 and X. By using Alu-element mediated PCR amplification and dot-blot hybridization with human alphoid or total human DNA as probes, 86 positive hybrids were identified and selected for further analysis. Nonisotopic fluorescence in situ hybridization (FISH) with human DNA in a set of eight hybrids demonstrated the presence of from one to eight human fragments per cell independent of irradiation dose. In contrast, a significant dose-dependent variation of fragment sizes was shown in the analysis of the 86 hybrids with markers previously mapped to 3p (seven markers) and to Xq (21 markers). Using the Xq27-28 region as a model, 40% of the hybrids generated at 5 krads or less were found to have retained fragments in the range of 3-30 Mb, 10% retained the whole chromosome arm, and the remaining 50% retained fragments of less than 2-3 Mb. The proportion of fragments of 3 Mb or larger decreased rapidly at higher irradiation doses and was very low (less than 6%) in hybrids generated at 25 krads. Upon further characterization, the 86 hybrids analyzed here will provide a mapping panel for the entire chromosomes 3 and X with an estimated resolution in the range of 1-2 Mb on average, a size range amenable to PFGE and YAC contig mapping.

Spontaneous and induced chromosome aberrations in Balkan endemic nephropathy.

The aim of the present study was to investigate the spontaneous aberrations and chromosome breakages induced by X-rays and folic acid deficiency. In patients with Balkan endemic nephropathy (BEN) a higher frequency of spontaneous aberrations and chromosome lesions in medium TC 199 and radiation induced breakages were found compared with the healthy individuals. In BEN patients the 3q25 band was most frequently involved in the aberrations. These results support the idea that 3q25 may play a specific role and be a marker for BEN. Three of the additional five bands with increased frequencies of lesions in BEN patients contain oncogenes: 1q36-c src, 3p25-raf-1, and 6q23-myb. The frequent association of BEN and cancer can be explained by the chromosomal hypothesis of oncogenesis.